Angiogenesis is the growth of new blood vessels from the existing vasculature. It is implicated in many diseases and disciplines, including heart disease where it plays an important role in repair of ischemic tissue following myocardial infarction. Heart disease is the second leading cause of death in the United States. Hope for effective angiogenesis therapies in ischemic heart disease has been dampened by a series of mostly disappointing clinical trials. Despite the importance of angiogenesis, there is a lack of effective angiogenesis assays and this is hampering progress in developing angiogenic therapeutics. While there exist a number of vitro and in vivo screening assays which can provide information about specific, narrow aspects of angiogenesis, it is often very difficult to interpret how the results of these assays pertain to the physiological relevant 3D microscopic tissue level where angiogenesis actually manifests. In particular, there is a conspicuous lack of assays that provide direct information about capillary morphology and function in situ even though this is ultimately the most important aspect of angiogenesis. This proposal is for the development of a high throughput 3D angiogenesis assay at the in situ tissue level. It accomplishes this by employing high speed 3D microscopic imaging, multispectral detection, advanced tissue labeling protocols, and software for high content image analysis. As a first application we will apply this to study the angiogenic response to fibroblast growth factor (bFGF) in myocardial ischemia. We believe this assay will become a valuable tool for basic researchers and provide crucial information in the late drug development process to pharmaceutical companies on whether to continue the development of a drug candidate. PUBLIC HEALTH RELEVANCE: This proposal describes the development of a new angiogenesis assay which we apply to the study of heart disease. Its key innovations are highly detailed 3D images of the microvascular, and the ability to this back to tissue physiology, composition and biochemical state. It has the strong potential to reduce the cost of doing angiogenesis assays in animal models as well as offer high physiological relevance and advance the use of angiogenesis therapies for the treatment of heart disease.